Immobilization methods encapsulation

Wei, Y., Xu, J., Feng, Q., Lin, M., Dong, H., Zhang, W.-J. and Wang, C. (2001) A novel method for enzyme immobilization direct encapsulation of add phosphatase in nanoporous silica host materials. Journal of Nanoscience and Nanotechnology, 1, 83-93. 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Various lipase immobilization methods were tested with different silica matrices, and the immobilized enzyme samples were examined by morphologic, physicochemical, and biochemical characterization methods. The results allowed correlation of the activity-coupling yield of different immobilization methods in relation to the incorporation of lipase in the silica gels and showed that the most active biocatalyst resulted from the encapsulation of commercial CRL in the presence of PEG. 

Physical immobilization methods do not involve covalent bond formation with the enzyme, so that the native composition of the enzyme remains unaltered. Physical immobilization methods are subclassified as adsorption, entrapment, and encapsulation methods. Adsorption of proteins to the surface of a carrier is, in principle, reversible, but careful selection of the carrier material and the immobilization conditions can render desorption negligible. Entrapment of enzymes in a cross-linked polymer is accomplished by carrying out the polymerization reaction in the presence of enzyme the enzyme becomes trapped in interstitial spaces in the polymer matrix. Encapsulation of enzymes results in regions of high enzyme concentration being separated from the bulk solvent system by a semipermeable membrane, through which substrate, but not enzyme, may diffuse. Physical immobilization methods are represented in Figure 4.1 (c-e). 

Figure 4.1. Enzyme immobilization methods, (a) Nonpolymerizing, (b) cross-linking, (c) adsorption, (d) entrapment, and (e) encapsulation.

The behavior of immobilized enzymes differs from that of dissolved enzymes because of the effects of the support material, or matrix, as well as conformational changes in the enzyme that result from interactions with the support and covalent modification of amino acid residues. Properties observed to change significantly upon immobilization include specific activity, pH optimum, Km, selectivity, and stability.23 Physical immobilization methods, especially entrapment and encapsulation, yield less dramatic changes in an enzyme s catalytic behavior than chemical immobilization methods or adsorption. The reason is that entrapment and encapsulation result in the enzyme remaining essentially in its native conformation, in a hydrophilic environment, with no covalent modification. 

Improve liposome encapsulation and investigate additional immobilization methods... 

Immobilization can be achieved by adsorption or covalent fixation of the biocatalyst to a solid support (e.g. surface-modified polymer or glass beads), by entrapment or by encapsulation in gel beads (e.g., agarose, polyacrylamide, alginate, etc.). Hundreds of immobilization methods have been described and reviewed in the literature [83-89], but only a limited set of methods has found real technical applications. The first large-scale applications of immobilized enzymes were established for the enantioseparation of D- and L-amino acids by Chib-ata, Tosa and co-workers at Tanabe Seiyaku Company. The Japanese achievements in the large-scale application of immobilized systems are very well documented in an excellent multi-author publication edited by Tanaka, Tosa and Kobayashi [90] (see also section 7). Some enzyme suppliers sell important industrial enzymes not only in the free form (solution or powder) but also immobilized on solid supports. 

The encapsulation of enzymes in a sol gel matrix is an immobilization method in which the enzymes are incorporated into hybrid organic-inorganic hydrophobic materials (alkylsilanes) [13]. The sol-gel process can easily be recognized because it is the synthesis route in which at a certain time, there is the transition from the sol to the gel system. In contrast to the conventional methods, which require high temperatures for the fusion of vitreous silica, the sol-gel technique uses low temperatures for the hydrolysis and... 

The drawbacks of previous methods can be overcome by a combining two of any immobilization methods (Kanwar et al., 2004 Yadav and Jadhav, 2005 Zarcula et al., 2009). Ursoiu et al. (2011) deposited immobilized C. antarctica lipase B via sol-gel entrapment on support material. Yadav and Jadhav (2005) tested the preimmobilization of C. antarctica lipase B on hexagonal mesoporous silica using physical adsorption, followed by encapsulation in calcium alginate beads, which resulted in a reusable lipase with no leaching even after the fourth reuse. 

Very few examples have been described for the non-covalent immobilization of chiral porphyrin complexes (Fig. 26). In the first case, the porphyrin-dichlororutheninm complex was encapsulated in silica, which was prepared around the complex by a sol-gel method [78], in an attempt to prevent deactivation observed in solution in the epoxidation of different alkenes with 2,6-dichloropyridine N-oxide. In fact, the heterogeneous catalyst is much more active, with TON up to 10 800 in the case of styrene compared to a maximum of 2190 in solution. Enantioselectivities were about the same imder both sets of conditions, with values aroimd 70% ee. 

Immobilization of chiral complexes in PDMS membranes offers a method for the generation of new chiral catalytic membranes. The heterogenization of the Jacobsen catalyst is difficult because the catalyst loses its enantioselectivity during immobilization on silica or carbon surfaces whereas the encapsulation in zeolites needs large cages. However, the occlusion of this complex in a PDMS matrix was successful.212 The complex is held sterically within the PDMS chains. The Jacobsen catalyst occluded in the membrane has activity and selectivity for the epoxidation of alkenes similar to that of the homogeneous one, but the immobilized catalyst is recyclable and stable. 

Common to all encapsulation methods is the provision for the passage of reagents and products through or past the walls of the compartment. In zeolites and mesoporous materials, this is enabled by their open porous structure. It is not surprising, then, that porous silica has been used as a material for encapsulation processes, which has already been seen in LbL methods [43], Moreover, ship-in-a-bottle approaches have been well documented, whereby the encapsulation of individual molecules, molecular clusters, and small metal particles is achieved within zeolites [67]. There is a wealth of literature on the immobilization of catalysts on silica or other inorganic materials [68-72], but this is beyond the scope of this chapter. However, these methods potentially provide another method to avoid a situation where one catalyst interferes with another, or to allow the use of a catalyst in a system limited by the reaction conditions. For example, the increased stability of a catalyst may allow a reaction to run at a desired higher temperature, or allow for the use of an otherwise insoluble catalyst [73]. 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

Recently, we proposed an alternative process for encapsulating biomacromolecules within PE microcapsules. This approach involves using nanoporous particles as sacrificial templates for both enzyme immobilization and PE multilayer capsule formation (Figure 7.2, route (I)) [66,67]. Unlike previous LbL encapsulation strategies, this approach is not limited to species that undergo crystallization, and is not dependent upon adjustments in electrostatic interactions within PE microcapsules to alter shell permeability characteristics. The salient feature of this method is that it is applicable to a wide range of materials for encapsulation. 

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